Viruses infect plants and animals including humans and microbes and amplify. Some are DNA viruses having DNA as a genome and others are RNA viruses having RNA as a genome; these viruses have different amplification mechanisms. Many viruses cause viral infection when infecting animals including humans. Viruses cannot increase by themselves, and can increase by infecting cells of other animals/plants/microbes and using the capability of the cells. Cells that a virus can infect for growth are called “host cells” for the virus. The type of host cells that a virus can infect for growth is dependent on the type of the virus.
Parvovirus is a small single-stranded DNA virus, and is an envelope-free icoshedral virus having a diameter as small as about 20 nm (Non Patent Literature 1). Parvovirus infects animals to cause a disease. Known examples of the disease include anemia due to simian parvovirus (SPV), cat enteritis/leucopenia/dystonia due to feline parvovirus (FPV), dog enteritis/myocarditis due to canine parvovirus (CPV), pig stillbirth due to porcine parvovirus (PPV), cow enteritis due to bovine parvovirus (BPV), goose enteritis/myocarditis due to goose parvovirus (GPV), and mouse enteritis/hepatitis due to minute virus of mice (MVM) in addition to infectious erythema, anemia, and arthritis that B19 parvovirus causes in humans (Non Patent Literatures 2 and 3). Parvovirus is important as a pathogen causing diseases in animals kept by humans, such as dogs and cats. When dogs are infected with canine parvovirus, they are known to experience enteritis as described above, develop severe diarrhea and vomiting, and die (Non Patent Literature 3). When cats are infected with parvovirus, they sometimes develop acute enteritis or leukopenia and also have the possibility of dying from a secondary infection, and when fetuses or newborn infants are infected with the virus, they may be damaged in the central nerve and the thymus to develop ataxia or die.
To prevent parvovirus infection, vaccines against parvovirus have been studied (Patent Literatures 1 and 2). To perform these studies, it is necessary to produce the virus for use. Many viruses can be grown and produced by culturing host cells and infecting the cells with the viruses. The production of a vaccine whose virus is attenuated or inactivated is achieved by the same procedure as that for virus production.
In the pharmaceutical industry, it is necessary to evaluate virus clearance (removal performance) of the production steps in order to assure no contamination of a pharmaceutical product of biological origin, such as a recombinant pharmaceutical product (a biopharmaceutical product) or an antibody pharmaceutical product, with virus (virus safety). Hence, the virus clearance of the individual step is measured by adding the virus to an intermediate pharmaceutical product before each step and quantitating the amount of the virus before and after the step. Particularly, porcine parvovirus (PPV) as one type of parvovirus is used at a high frequency in the virus clearance evaluation of a plasma derivative performed by the method described in the ICH (International Conference on Harmonisation of Technical Requirements for Registration of Pharmaceuticals for Human Use) guideline prescribed for the method of selecting the type of the virus used for the virus clearance evaluation of the producing steps of a biologics, and minute virus of mice (MVM) as a type of parvovirus is used at a high frequency in the virus clearance evaluation of a biopharmaceutical product. Thus, parvovirus is used at a high frequency for the virus clearance evaluation of the producing steps of a biologics.
There are a method using a laboratory animal, a method using hen's eggs, and a method using tissue culture/cultured cells to produce a virus (Non Patent Literature 4). The methods using a laboratory animal and hen's eggs have the disadvantage of high cost. The alternate method is a method using cultured cells (Non Patent Literature 5). Parvovirus is also produced by a method using cultured cells (Patent Literature 1).
To produce a virus such as parvovirus, a method is commonly performed which involves infecting a culture system of host cells with its seed virus and growing and recovering the virus. As used herein, the seed virus is referred to a small amount of the virus used at the beginning of virus growth deemed as “seed”. In conventional virus production, the timing at which host cells are infected with the seed virus is typically a stage at which the host cells have reached confluence to form a single layer state (Non Patent Literature 4 and Patent Literatures 3 to 6). In other words, typically, host cells are inoculated in a culture vessel and grown so that the host cells spread by growth on the full area of the bottom face of the culture vessel and then, a seed virus is inoculated; this is because when infectable cells are present at higher density, a system for a place in which more particles of the virus are produced is provided. Two to three days are typically required from the inoculation of the host cells in the culture vessel until their reaching a confluent state (Non Patent Literature 4). In the confluent state, the host cells are in a stationary phase and do not further grow. Thus, the conventional technology involves completing a growing culture step for host cells, starting virus infection in a culture environment in which the cells are not further grown, and producing the virus in the culture supernatant in parallel with the death of the host cells from the virus infection. Parvovirus is no exception in such a method; the virus was produced by a method involving infecting cells in a state of confluence (Non Patent Literatures 6 and 7) and the infectivity titer of the resultant parvovirus was 105 to 107 TCID50/mL. In the conventional culture system, parvovirus is added to host cells in a confluent state in which the number of cells is highest, and the added parvovirus grows in host cells and increases with the attendant death of the host cells. A culture supernatant can be recovered at a stage when the infectivity titer of parvovirus becomes highest to recover a parvovirus solution having a highest infectivity titer. The parvovirus obtained in the culture supernatant by this method is naturally recovered in a state suspended in the medium provided for the cell culture.
The removal of impurities is also performed for the parvovirus solution obtained as described above. The removal of impurities, such as cell debris, by low-speed centrifugation is carried out as the removing method. As methods for removing more impurities, there are also known cesium chloride density-gradient ultracentrifugation using an ultracentrifugation technique and a sucrose density-gradient ultracentrifugation technique (Non Patent Literature 8).